Studies on Antimicrobial activity of a metabolite produced by Streptomyces malaysiensis isolated from Termite Mound Soil

 

Uma Maheswari Kolipaka¹*, G. Girija Sankar²

¹Assistant Professor, Pharmaceutical Biotechnology Department, Narasaraopet, Guntur, A.P-522601

^2.Professor, A.U. College of Pharmaceutical Sciences, Visakhapatnam

 

ABSTRACT:

To discover novel secondary metabolites, our approach is to investigate unexplored regions of the world with the aim of isolating bioactive actinomycetes from the termite mounds soil sample. Actinomycetes are soil microorganisms. They are a type of bacteria. They are the same as each microorganism and fungi, and have characteristics linking them to both groups Actinomycetes square measure typically believed to be the missing biological process link between microorganism and fungi, however they need more characteristics in common with microorganism than they are doing fungi.13 actinomycetes and 3 fungal isolates were screened from the soil sample and out of them 6 actinomycetes showed anti microbial activity by agar-overlay method, Cross streaking method and Cup plate method. By the Agar overlay method we can assure that the isolate TMS 1A is having anti bacterial activity and anti fungal activity by measuring the inhibition zone diameter as 40mm and 35mm respectively. Cross streaking method of the active compound obtained from isolate TMS 1A shows that it is having antimicrobial activity against Gram +ve, Gram -ve bacteria and fungi. Cup plate assay of the active compound obtained from isolate TMS 1A shows that it is having anti bacterial activity. Results from anti microbial activity revealed that our isolate TMS 1 08 09 2010 1A was found to have broad spectrum of anti microbial activity on both (Gram + ve and Gram – ve) bacteria and fungi. Further investigation is required to extract the Bioactive metalbolites from Streptomyces malaysiensis.

 

 

 

INTRODUCTION:

Aim and objectives of the work:

To discover novel secondary metabolites, our approach is to investigate unexplored regions of the world with the aim of isolating bioactive actinomycetes from the termite mounds soil sample. The aim of the present study is to optimize to the fermentation for proliferating the antibiotic production by soil actinomycete exhibiting promising antibacterial and antifungal activities.

 

 

REVIEW OF LITERATURE:

Amira Al-Tai, Bongcheol Kim, Seung, Bum Kim, Gilson, Paulo, Manfio and Michael Goodfellow. The taxonomic position of a streptomycete strain isolated from Malaysian soil was established using a polyphasic approach. The organism, designated strain ATB-11^T, was found to have chemical and morphological properties consistent with its classification in the genus Streptomyces. An almost complete 16S rRNA gene (rDNA) sequence determined for the test strain was compared with those of previously studied streptomycetes by using two treeing algorithms. The 16S rDNA sequence data not only supported classification of the strain in the genus Streptomyces but also showed that it formed a distinct phyletic line At maturity, the aerial hyphae of strain ATB-11^T differentiated into tight spira chains of rugose, cylindrical spores. The organism was readily distinguished from representatives of validly described Streptomyces species with rugose spores by using a combination of phenotypic features. It is proposed, therefore, that strain ATB-11^T be classified in the genus Streptomyces as Streptomyces malaysiensis sp. nov.

 

Jinhua Cheng, Seung Hwan Yang, One hundred and sixty two actinomycete strains isolated from Brazilian soils were screened for xylanase activity, according to the size of the hydrolysis zones observed in oat spelts xylan agar plates. The strain AMT-3, later identified as Streptomyces malaysiensis, was selected as the best producer. In subsequent shake flasks fermentations using growth media contanning 1% (w/v) of either birchwood, or oat spelts xylan, plus organic nitrogen and salts, high endo-β-1,4-xylanase titres (EC 3.2.1.8) (116 U ml⁻¹) were observed in the larchwood medium within 6 days. This is the first report concerning xylanase production by streptomyces malaysiensis, which has been recently described as a new species.

 

Sasikumar Arunachalam Palaniyandi, Jung Sun Han, Azalomycin F complex is an antifungal substance produced by Streptomyces malaysiensis MJM1968 isolated from agricultural soil. MJM1968, an actinomycete showing potent antifungal activity, was isolated and identified by 16SrDNA sequence analysis as Streptomyces malaysiensis. MJM1968 showed strong antifungal activity on phytopathogenic fungi such as Fusarium oxysporum, Rhizoctonia solani, Cladosporium cladosporioides, Fusarium chlamydosporum, Colletotrichum gloeosporioides KACC 40693, Alternaria mali KACC 40026, and Pestalotia spp. KACC 40501, in vitro. An antifungal compound was isolated from culture filtrate of strain MJM1968 by a series of chromatographic methods. Treatment of agricultural soil with strain MJM1968 mycelia reduced the native fungal population by more than 60% after 14 days. Treatment of soil with 10 mg/mL partially purified extract lowered native fungal density by more than 80% after 14 days. The compound, which exhibited antifungal activity in a broad range of pH and temperature, was identified as azalomycin F complex by ¹H and¹³C NMRs. This is the first report on the isolation of azalomycin F complex from S. Malaysiensis, demonstrating a broad-spectrum suppression of fungal pathogens in agricultural soil.

 

Yasuhiro Igarashi1, Yuki Tanaka1, Streptomyces malaysiensis AMT-3, isolated from a Brazilian cerrado soil, showed proteolytic activities detected by gelatin-sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The optimum proteinase production was obtained when using 2.5% wheat bran and 0.1% yeast extract in the culture medium, after 5 days incubation at 30 degrees C. The enzymatic complex degraded gelatin optimally at pH 7.0, and under these conditions eight proteolytic bands (four serine-proteinases and four metaloproteinases), ranging from 20 to 212 kDa, were detected on the culture supernatant filtrates. In addition, a 35-kDa proteinase was thermostable at 60 degrees C for 120 min. These results point out to the applicability of gelatin zymograms in the characterization of crude enzymatic complexes. According to our results, this enzymatic complex could be used for biotechnological applications.

 

MATERIALS:

Table 1 Equipments used:

Name of the Equipment	Name of the Manufacturer
Autoclave	REMI Instruments Ltd., Mumbai
Hot air oven	BIOMED INC.
Incubator maintained at 37°C	REMI Instruments Ltd., Mumbai
Orbital Rotary Shaker	REMI Instruments Ltd., Mumbai
Centrifuge	REMI Instruments Ltd., Mumbai
Centruguge tubes	Tarsons Products Pvt. Ltd., Kolkata
Rotary Evaporator	Heidolph (Labrota 4000)
Laminar Airflow Unit	KLENZAIDS
Analytical weighing Balance	Shimadzu (aux 220),Japan
Millipore	Quantum – IX
Cooling Centrifuge	REMI Instruments Ltd., Mumbai
Cooling Incubator	REMI Instruments Ltd., Mumbai
Microtips (200 – 1000µL)	Tarsons products Pvt. Ltd., Kolkata
Micropipette (10 – 1000 µL)	Tarsons products Pvt. Ltd., Kolkata
Deep freezer	Cell Frost (CF – 200)
UV- Spectrophotometer	Elico
Trinocular microscope	Labomed – CXR3

 

Table 2 Glassware used:

Borosil

Test tubes, boiling tubes, conical flasks, beakers, petriplates, measuring cylinders, funnels, separating funnels, loops, Borers, pH papers

 

Table 3 Organic solvents used:

Name of the organic solvent	Name of the Manufacturer
Ethyl acetate	Merck Limited, Mumbai
Ethanol	Merck Limited, Mumbai
Methanol	Merck Limited, Mumbai

 

Table 4 Chemicals used:

Name of the chemical	Name of the Manufacturer
Peptone	Qualigens fine Chem. Mumbai
Sodium chloride	Himedia Lab., Mumbai
Meat extract	Himedia Lab., Mumbai
Nutrient Agar	Himedia Lab., Mumbai
Soluble starch	Himedia Lab., Mumbai
Hydrochloric acid solution 0.1M	Qualigens fine Chem. Mumbai
Potassium nitrate	Qualigens fine Chem. Mumbai
Sodium hydroxide solution 0.1M	Qualigens fine Chem. Mumbai
Hydrated Magnesium sulphate	Himedia Lab., Mumbai
Calcium carbonate	Qualigens fine Chem. Mumbai
Ferrous sulphate	Himedia Lab., Mumbai
Soya bean meal	Himedia Lab., Mumbai
Dextrose	Qualigens fine Chem. Mumbai
Glycerol	Qualigens fine Chem. Mumbai
Sodium nitrate	Qualigens fine Chem. Mumbai
Zinc sulphate	Himedia Lab., Mumbai
Di potassium hydrogen phosphate	Himedia Lab., Mumbai
Yeast extract powder	Qualigens fine Chem. Mumbai

Table 5 List of test organisms used:

NCIM: National Collection of Industrial Microorganisms;  MTCC: Microbial Type Culture Collection

 

 

EXPERIMENTAL METHODS:

Isolation of actinomycetes from termite soil samples:

Sample collection:

Sample was collected at AU Out gate, Andhra University, AU college of Pharmaceutical Sciences, Visakhapatnam. By using sterile spatula 5gms of soil was collected into self sealed cover by taking at different spots on the termite mound. The composition of the screening medium used for the screening of the soil sample is given Table.

 

Table 6 Starch Casein Agar Medium:

 

Steps involved in the screening of soil sample:

·      5gms of collected sample was inoculated into 50 ml of sterile water aseptically and kept on rotary shaker for half an hour.

·      Then 9ml of sterile water was sterilized previously and into it 1ml of the sample was inoculated into it.

·      Sterile dilutions from 10^-1 to 10^-3 were inoculated.

·      Antibiotic solutions of Rifampicin and Cycloheximide were prepared and each 1 ml were transferred to the sterilized starch casein agar medium and 1 ml of serially diluted sample into it and poured into previously sterilized petriplates and kept for solidification.

·      Then observe the growth of colonies of actinomycetes on the petri plates.

 

13 actinomycetes colonies are isolated from petri plates, out of 13 isolates one was found to be consistent and reproducible with respect to antibiotic production and was selected for submerged fermentation studies.

 

 

Preliminary testing for antibiotic production of these isolates was done using production medium (PM 6). Antimicrobial activity was tested following cup plate method.

 

Inoculum preparation:

Bacterial inoculums preparation:

A pure colony of the test organism was taken using a sterile loop and transferred into tubes having a sterile nutrient broth and incubated with shaking at 35°C-37°C until the visible turbidity was equal to that of the 0.5 McFarland standard.

 

Fungal inoculums preparation:

Three to seven days slant of fulgal culture on potato dextrose agar was taken and scrapped to form a suspension in sterile water. The mixture was wortexed and heavy particles were allowed to settle. The suspension was adjusted to 0.5 McFarland standards.

 

Inoculation procedure:

Within 15 minutes after adjusting the turbidity of the inoculums suspension, sterile cotton swab was dipped into the suspension. The swab was pressed firmly against the inside wall of the tube above the fluid level, the swab was rotated to remove excess liquid. The swab was streaked over the entire surface of the medium three times, rotating the plate approximately 60 degrees after each application to ensure an even distribution of inoculums. Finally all round the edge of the agar surface was swabbed.

 

Selection of micro organism:

MS01 1A which was earlier isolated in the Pharmaceutical Division of Biotechnology University College of Pharmaceutical Sciences, Andhra University, Visakhapatnam and selected for the optimization studies.

 

Determination of Antimicrobial activity:

The antimicrobial activity of the MS01 1A strain was observed by performing the following tests.

 

 

 

Cross streak method:

The preliminary screening of the isolates can be determined by the cross streak method. In this method fully sporulated isolates which are previously subcultured are taken and crossly streaked over the solidified YEME agar plates and kept for incubation at 27°C for 5 to 7 days. Then observe the growth of the actinomycetes sporulated well or not. Then 24hrs cultures of the Gram +ve and Gram -ve bacteria are subcultured and crossly streaked over the sporulated plate of the actinomycetes and incubated at 37°C for 24 to 48 hrs. Same as that subcultured fungi also crossly streaked over the sporulated plate of the actinomycetes and incubated at 28°C for 48 hrs.  And observe the anti microbial activity.

 

Table 7 List of test Organisms used:

Gram positive	Staphylococcus aureus, NCIM-2079, Basillus Subtilus.
Gram negative	Escherichia coli, NCIM-2065, Klebsiella Pneumonia.
Fungi	Candida albicans

 

Agar overlay method:

In this method the isolates are sub cultured and then the sporulated cultures are stabbed on the solidified YEME agar medium plates. After sporulation of isolates in the plates where we stabbed then nutrient agar medium of 0.8% agar concentration was prepared and sterilized and inoculated with 24hrs culture of Gram +ve and Gram -ve bacteria and sabouraud’s agar medium for fungi is prepared and inoculated in it and poured over the stabbed plated. Then the results were observed after 24hrs for bacteria and after 48hrs for fungi. Then the zones of inhibition were measured after 24hrs of incubation at 37°C for bacteria and 48hrs of incubation at 28°C for fungi.

 

Table 8 List of test organisms used:

Gram positive	Staphylococcus aureus, NCIM-2079
Gram negative	Escherichia coli, NCIM-2065
Fungi	Aspergillus niger

 

Agar diffusion method/cup plate method:

The active compound obtained after extraction and evaporation is tested for its antimicrobial activity. Nutrient agar medium was prepared and sterilized and inoculated with the organism is poured into the petri plates under aseptic conditions. Cups were made and into these, the active compound is added using a micropipette and then kept for refrigeration for half an hour to improve the diffusion. Then the plates were kept for incubation at 37°C, 24 hrs in case of bacteria and 28°C, 48 hrs in case of fungi.

 

Taxonomy of the isolate:

The taxonomy of the isolate includes the morphology and the phenotypic characteristics.

Morphology:

The highly sporulated culture of isolate was required for morphological identification. Into the sterile petri plate, YEME medium was taken as a thin film and solidify it and then sterile water was taken and inoculated with the fresh sporulated culture spread the inoculums over the YEME media solidified. Then kept for incubation at 28°C and observe the growth of spores in the plate next day onwards. Finally check the fragmented spores of isolate under the microscope for identification.

 

Phenotypic characteristics:

The isolate MS01 1A was sent for identification of phenotypic characteristics to IMTECH Chandigarh.

 

Fermentation:

The fermentation process involves preparation of inoculums medium and preparation of production medium (PM)

 

Preparation of inoculums medium:

5ml of sterile water was added to SCA slant and scraped with sterile loop and inoculated to 50ml of sterile medium of MM (Modified Medium) under the aseptic conditions then the flasks are incubated at 28°C for 2 days on rotary shaker.

 

Preparation of the production medium:

10% of the inoculums medium was added to the production medium (PM7). And incubated on a rotary shaker (180rpm) at 28°C for 7 days of the submerged fermentation cycle.

 

Extraction of active compounds:

The sample were withdrawn at the end of the fermentation cycle centrifuged and the supernatant was extracted with ethyl acetate (1:3) ratio. And further concentration of the crude extract was done by using rotary evaporator. Then the compound was tested for antibacterial and anti fungal activity by agar diffusion met

 

Optimization studies:

Selection of suitable inoculums medium:

To minimize the time lag in fermentation process inoculums medium similar to selected production medium were used. The composition of the suitable inoculum medium is shown in the table 11.

 

Table 9

10%  Inoculum was added to above media and incubated on a rotary shaker (120rpm) at 28°C for 5 days. Samples were withdrawn at the end of the fermentation cycle i.e 120hrs, centrifuged and assayed for antibacterial and antifungal activity as per the general procedure of cup plate assay.

 

RESULTS AND DISCUSSION:

Sample collection:

The soil sample from termite mounds was collected from AU out gate, AU college of Pharmaceutical sciences, Andhra university, Visakhapatnam and screened for the isolation of actinomycetes. The following figure denotes a termite mound.

 

Fig No. 1 Termite mound

 

Screening of the soil sample:

13 actinomycetes and 3 fungal isolates were screened from the soil sample and out of them 6 actinomycetes showed anti microbial activity by agar-overlay method

 

Fig No. 2 Antimicrobial activity of some of the actinomycetes isolates by agar overlay method

 

 

Selection of suitable production medium:

To select the best production medium for antibiotic production 8 different production medium are evaluated. SS01 1A is inoculated into 8 different mediums and yield of antibiotics produced are determined by cup plate assay. The table 10 shows the antimicrobial activities of  the isolates in different production media.

 

Table 10

Isolate	Medium	Inhibition zone diameter (mm)
		Test organisms
		Staphalococus aureus	Escherisia coli	Aspergillus niger
TMS 1 A	PM 1	25	20	25
	PM 2	27	24	20
	PM 3	30	28	25
	PM 4	35	40	38
	PM 5	30	35	30
	PM 6	36	35	30
	PM 7 (Modified PM 6)	45	40	40
	YEME broth	40	38	38

 

The modified PM 6 medium shows high yield of antibiotic production by showing high antimicrobial activity.

 

Determination of Anti microbial activity:

Agar Overlay Method:

In this method as we discussed earlier zones of inhibition were measured after 24 hrs of incubation at 37°C for bacteria and 48 hrs of incubation at 28°C for fungi for the selected isolate TMS 1A. antimicrobial activity of the selected isolate TMS 1A shown in the following fig.

 

Fig No. 3 Antibacterial activity of TMS 1A by Agar Overlay

Method

 

By the Agar overlay method we can assure that the isolate TMS 1A is having anti bacterial activity and anti fungal activity by measuring the inhibition zone diameter as 40mm and 35mm respectively.

 

Cross Streking method:

In this method as we discussed earlier that the anti microbial activity of the compound isolated from the isolate TMS 1A was as follows:

 

Fig No. 4.4 Antibacterial activity of 1A against Gram +ve Bacteria

 

Fig No.4.5 Antibacterial activity of 1A against Gram –Ve Bacteria

 

Fig No.4.6 Antifungal activity of 1A against Candida albicans

 

Cross streaking method of the active compound obtained from isolate TMS 1A shows that it is having antimicrobial activity against Gram+ve, Gram-ve bacteria and fungi.

 

Cup plate assay method:

In this method as we discussed before that the anti microbial activity of the compound isolated from the isolate TMS 1A was as follows:

 

Fig No.4.7 Cup plate of assay of the isolate TMS 1A

 

Cup plate assay of the active compound obtained from isolate TMS 1A shows that it is having anti bacterial activity.

 

Taxonomy of the promising isolate:

Morphology of the isolate:

Under the microscopy it was observed as fragmented net work of mycelium having spores.

 

Fig No. 4.8 The microscopic image of the isolate TMS1A

 

Phenotypic Characterization of the selected isolate TMS1A:

Microscopic observation indicates that the isolated Organism has similar features to that of actinomycetes. So it is further confirmed by IMTECH Chandigrah. As Actinomycetes.

 

Summary:

The summary of my work, initially subculturing of the actinomycetes isolates from the stock cultures. Then the isolates are tested by preliminary screening procedure by cross streak and Agar overlay method for determining the anti microbial activity. After preliminary screening the results indicates that 6 isolates having the antimicrobial activity. Out of that 1A is having highest antimicrobial activity so we have selected 1A for the further studies. Selection of suitable inoculum medium and selection of suitable production medium for the growth of 1A is optimized. After optimization of the media PM 7 medium is showing highest activity against cup plate assay, so PM 7 is considered as the best medium for the production of antibiotics by 1A. The morphological features of 1A under the trinocular microscope reveals that it may belongs to genera Streptomyces and phenotypic characteristics are revealed by the IMTECH Chandigarh report which they have sent. And separation of active compound is under further studies.

 

CONCLUSIONS:

The conclusions of my project work includes:

·      A total of 13 Actinomycetes were subcultured from stock cultures.

·      From the selected sample TMS 1 08 09 2010, 13 actinomycetes isolates were isolates and screened for activity where 6 actinomycetes were found to be active against test organisms.

·      From the termites soil sample TMS 1 08 09 2010, 1A was selected from the inhibition zone diameters for further studies.

·      Results from anti microbial activity revealed that our isolate TMS 1 08 09 2010 1A was found to have broad spectrum of anti microbial activity on both (Gram + ve and Gram – ve) and fungi.

·      Phenotypic characterization of this isolate revealed that it belongs to the genus streptomyces and identified it as to be Streptomyces malaysiensis.

·      Parameters like inoculums medium production medium were optimized to get high yield of the bioactive molecules and found that modified PM 6 showed good yield of the bioactive molecule.

 

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Received on 18.12.2018        Modified on 10.02.2019

Accepted on 05.03.2019        © RJPT All right reserved